LOCUS       sequence-146280-        4097 bp    DNA     circular SYN 15-APR-2025
DEFINITION  fission yeast epitope tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4097)
  AUTHORS   Amelina H, Moiseeva V, Collopy LC, Pearson SR, Armstrong CA, Tomita 
            K
  TITLE     Sequential and counter-selectable cassettes for fission yeast.
  JOURNAL   BMC Biotechnol. 2016 Nov 8;16(1):76.
  PUBMED    27825338
REFERENCE   2  (bases 1 to 4097)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "BMC 
            Biotechnol."; date: "2016-11-8"; volume: "16(1)"; pages: "76"
COMMENT     Sequence Label: pNX3a-FL3
FEATURES             Location/Qualifiers
     source          1..4097
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     43..61
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(99..121)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     221..240
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        321..339
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     321..338
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             412..435
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             439..486
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     terminator      516..703
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(571..595)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            762..2118
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        762..1105
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1106..1915
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1173..1192)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1921..2118
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2177..2194)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2348..2936)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2428..2447)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3107..3898,3899..3967))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3730..3749
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3968..4072)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
       61 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
      121 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
      181 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
      241 ctgagagtgc accatatgga catattgtcg ttagaacgcg gctacaatta atacataacc
      301 ttatgtatca tacacatacg atttaggtga cactatagaa cgcggccgcc agctgaagct
      361 tcgtacgctg caggtcgacg gatccccggg ttaattaacg ctagcatggg tgactacaag
      421 gacgacgatg acaagggaga ttacaaagat gacgacgata aggactacaa ggacgacgat
      481 gacaagtcta gatgaggcgc gccacttcta aataagcgaa tttcttatga tttatgattt
      541 ttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt gactcttagg
      601 ttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg ttgctttctc
      661 aggtatagta tgaggtcgct cttattgacc acacctctac cggcagatcc gctaggagat
      721 ctgtttagct tgcctcgtcc ccgccgggtc acccggccag cgacatggag gcccagaata
      781 ccctccttga cagtcttgac gtgcgcagct caggggcatg atgtgactgt cgcccgtaca
      841 tttagcccat acatccccat gtataatcat ttgcatccat acattttgat ggccgcacgg
      901 cgcgaagcaa aaattacggc tcctcgctgc agacctgcga gcagggaaac gctcccctca
      961 cagacgcgtt gaattgtccc cacgccgcgc ccctgtagag aaatataaaa ggttaggatt
     1021 tgccactgag gttcttcttt catatacttc cttttaaaat cttgctagga tacagttctc
     1081 acatcacatc cgaacataaa caaccatggg taaggaaaag actcacgttt cgaggccgcg
     1141 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     1201 gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct
     1261 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     1321 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     1381 atggttactc accactgcga tccccggcaa aacagcattc caggtattag aagaatatcc
     1441 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     1501 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc
     1561 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     1621 tgttgaacaa gtctggaaag aaatgcataa gcttttgcca ttctcaccgg attcagtcgt
     1681 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     1741 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     1801 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     1861 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcagt
     1921 actgacaata aaaagattct tgttttcaag aacttgtcat ttgtatagtt tttttatatt
     1981 gtagttgttc tattttaatc aaatgttagc gtgatttata ttttttttcg cctcgacatc
     2041 atctgcccag atgcgaagtt aagtgcgcag aaagtaatat catgcgtcaa tcgtatgtga
     2101 atgctggtcg ctatactgct gtcgattcga tactaacgcc gccatccagt ttaaacgagc
     2161 tcgaattcta ggcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     2221 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     2281 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     2341 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     2401 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     2461 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     2521 cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg
     2581 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     2641 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     2701 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     2761 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     2821 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     2881 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     2941 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     3001 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     3061 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     3121 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     3181 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     3241 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     3301 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     3361 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     3421 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     3481 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     3541 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     3601 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     3661 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     3721 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     3781 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     3841 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     3901 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     3961 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     4021 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     4081 cccgaaaagt gccacct
//
